Heart failure is a leading cause of death and indication for cardiac transplantation in the US. Non- ischemic idiopathic dilated cardiomyopathy (iDCM ), an important cause for heart failure, can arise from mutation in many different genes. Sequence analyses of iDCM genes often yield data with uncertain clinical significance due to a lack of knowledge of the full genetic architecture of iDCM and the significance of different gene variants. In this proposal we will sequence and analyze 105 candidate genes in three iDCM populations to define pathogenic genes and variants. We will harness cardiomyocytes differentiated from iPS cells (iPS-CM) to functionally validate gene variant classification and to explore the transcriptional responses to iDCM mutations. Through these studies we aim to a) Determine the allelic spectrum, including loss of function and missense variants that cause iDCM; b) Determine if the genetic architecture of iDCM varies in patients with different ancestries and c) Determine if iDCM genes perturb cardiac biology by altering shared or distinct molecules and pathways. These studies are predicated on successes achieved during the prior funding period. Under the aegis of this grant we demonstrated that truncating titin (TTNtv) cause ~13% unselected iDCM, 22% end- stage iDCM, and 25% severe, familial iDCM. Loss-of-function variants in ~20 other iDCM genes contribute to an additional 10% of the burden of iDCM. We will extend these studies to characterize iDCM genes and variants in patients with non-EU ancestry. Our sequencing data also revealed vast numbers of nonsynonymous variants that encode missense alleles. We will improve our interpretation of these rare missense alleles by harnessing robust bioinformatic strategies that incorporate large sequencing datasets to predict deleterious missense iDCM variants; these will be validated through functional analyses. To improve mechanistic understanding of iDCM we will capitalize on our success in unbiased transcriptional profiling of human and mouse iDCM tissues and iPS-CM that carry iDCM mutations. By defining the pathways and mechanisms that lead to iDCM we hope to identify therapeutic targets that can attenuate progression of disease to heart failure. Our specific aims are to: 1. Expand the genetic architecture of iDCM by studying patients with EU and non-EU ancestry 2. Construct iPS-CM with iDCM gene variants and assess pathogenicity by functional analyses 3. Produce and analyze selected iDCM gene mutations in vivo